Pharmaceutical compositions for the treatment of helicobacter pylori-associated disorders

ABSTRACT

The invention relates to pharmaceutical compositions and methods for treating and/or preventing  Helicobacter pylori -associated disorders, particularly disorders of the gastrointestinal tract. The pharmaceutical compositions comprise as active ingredient a therapeutically effective amount of a compound which inhibits the growth-enhancing effect of gastrin on  H. pylori . The active ingredient may specifically be a compound which is capable of inhibiting gastrin uptake by  H. pylori , and/or which is an antagonist of the human or  H. pylori  gastrin receptor.

RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/IL99/00335, which designated the United States and was filed onJun. 17, 1999, published in English, which claims the benefit of U.S.Provisional Application No. 60/147,195 filed on May 20, 1999 and U.S.Provisional Application No. 60/089,770 filed on Jun. 18, 1998. Theentire teachings of the above applications are incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The invention relates to pharmaceutical compositions for thetreatment and/or prevention of Helicobacter pylori-associated disordersand to methods of treating such disorders in patients in need thereof.

BACKGROUND OF THE INVENTION

[0003]Helicobacter pylori (H. pylori) infection is associated withseveral benign and malignant human diseases [Dooley CP, et al., N Engl JMed (1989) 321:1562-1566; Carrick J, et al., Gut (1989) 30:790-797;Nomura A, et al., N Engl J Med (1991) 325:1132-1136 [see comments];Nomura A, et al., Ann Intern Med (1994) 120:977-981 [see comments];Zucca E, et al., N Engl J Med (1998) 338:804-810]. However, symptomaticinfection is only the tip of the iceberg, the majority of infectedindividuals remain asymptomatic. Moreover, if untreated, infection maylast for decades [Peterson W L and Harford W V, (1991) 86:671-675],representing a successful host-parasite relationship. This favorableinteraction is reflected in a high prevalence of infection rangingbetween 50% in developed to 90% in developing countries [Graham D Y, etal. Dig Dis Sci (1991) 36:1084-1088; Taylor D N and Blaser M J,Epidemiol Rev (1991) 13:42-59].

[0004]H. pylori resides within the mucous layer of the human gastricmucosa. Due to extremely low pH, the stomach is a hostile environment tomost other microorganisms. The ability of H. pylori to flourish in thestomach has been attributed to protective mechanisms such as itsproduction of urease, protecting the bacterium from gastric acidity bycreating a basic microenvironment [Taylor D N and Blaser M J, EpidemiolRev (1991) 13:42-59]. However, it has now been reasoned, that H. pylorimight have evolved a way to gain growth advantage in this particularniche of the stomach, possibly by exploiting a gastric factor. A logicalcandidate would be one that is upregulated by H. pylori infection.

[0005] One such factor is the gastric hormone gastrin. Gastrin isproduced as a prohormone by G cells located within the gastric antrum.The prohormone is later processed to shorter peptides, the most abundantof which is 17 amino acids long, termed gastrin 17 (G17) [Eaton K A, etal, Infect Immun (1991) 59:2470-2475]. The major role attributed togastrin within the gastric tissue is the regulation of acid secretion.Following infection, gastrin levels are found to be consistentlyelevated and normal physiologic negative feedback control of secretionis lost. Further, following H. pylori eradication, gastrin levels arereduced and normal feedback control of gastrin secretion is restored[Graham D Y, et al., Am J Gastroenterol (1990) 85:394-398; El-Omar E, etal., Gut (1993) 34:1060-1065 [see comments]; Konturek J W, et al, Gut(1995) 37:482-487].

[0006] Interest in the changes of gastrin secretion and control havebeen directed to its role in acid production, and the resulting pepticpathology. However, the present work was focused on the possibleinteractions between gastrin and H. pylori.

[0007] As will be shown hereafter, gastrin positively stimulates thegrowth of H. pylori, a finding which further exemplifies the successfuladaptation of H. pylori to the human host, and provides the basis forthe present invention.

SUMMARY OF THE INVENTION

[0008] The present invention relates to pharmaceutical compositions forthe treatment and/or prevention of H. pylori-associated disorderscomprising as active ingredient a therapeutically effective amount of acompound which inhibits the growth-enhancing effect of gastrin on H.pylori, particularly H. pylori-associated gastrointestinal disorders,such as H. pylori-associated gastric and/or duodenal peptic diseases.The pharmaceutical compositions of the invention may optionally furthercomprise pharmaceutically acceptable carriers, adjuvants or diluents.

[0009] The active ingredient in the pharmaceutical compositions of theinvention may be a compound which is capable of inhibiting gastrinuptake by H. pylori, particularly compounds which are competitiveinhibitors of gastrin uptake by H. pylori, or antagonists of the humanor H. pylori gastrin receptor. In preferred embodiments, the activecompound in the pharmaceutical compositions of the invention is apeptide. Preferred peptides are synthetic analogues of gastrin or of afragment of gastrin, preferably of G17, and most preferred are peptidescomprising the amino acid sequence: Trp-Met-Asp-PheNH₂, such aspentagastrin or cholecystokinin (CCK)-8.

[0010] In other embodiments the pharmaceutical compositions of theinvention may comprise as the active ingredient a non-peptidicantagonist of the human or H. pylori gastrin receptor.

[0011] In yet a further aspect, the invention relates to a method forthe treatment and/or prevention of H. pylori-associated disorders in apatient in need of such treatment, comprising administering to saidpatient a therapeutically effective amount of a compound which inhibitsthe growth-enhancing effect of gastrin on H. pylori or a therapeuticallyeffective amount of a composition according to the invention.

[0012] The method of the invention may be used for the treatment and/orprevention of H. pylori-associated gastrointestinal disorders.

[0013] Yet further, the invention relates to use of a compound whichinhibits the growth-enhancing effect of gastrin on H. pylori in thepreparation of a pharmaceutical composition for the treatment of H.pylori-associated disorders. In the use according to the invention, thesaid compound may be a synthetic analogue of G17, preferably comprisingthe amino acid sequence: Trp-Met-Asp-PheNH₂. Alternatively, the compoundmay be a non-peptidic antagonist of the human or H. pylori gastrinreceptor.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIGS. 1A to 1B H. pylori growth kinetics in the presence ofgastrin.

[0015] Bacteria were grown in liquid media, using microaerophilicconditions with increasing gastrin concentrations. Growth rate wasassessed by optical density (OD). T indicates time in hours.

[0016]FIG. 1(A) shows a representative growth curve of one isolate over48 hours.

[0017]FIG. 1(B) shows cumulated growth data of 5 different clinicalisolates with (3.7 pM) or without gastrin. Growth is compared at 0 h and48 h (Shown as a mean with SEM).

[0018]FIGS. 2A to 2F Gastrin growth stimulation is bacterial and peptidespecific.

[0019] Growth kinetic analysis of different bacteria with gastrin:

[0020]FIG. 2A: Growth kinetics analysis of C. jejuni with gastrin;

[0021]FIG. 2B:: Growth kinetics analysis of E. coli with gastrin;

[0022]FIG. 2C: Growth kinetics analysis of H pylori with the gastricpeptide somatostatm;

[0023]FIG. 2D: Growth kinetics analysis of H pylori with EGF.

[0024]FIG. 2E: Growth kinetics analysis of H. pylori with human gastrinreceptor agonist pentagastrin;

[0025]FIG. 2F: Growth kinetics analysis of H. pylori with human gastrinreceptor agonist CCK-8.

[0026] Bacteria were grown using liquid media and growth rate assessedby optical density (OD). T indicates time in hours. Except the aerobicconditions used for the growth of E. coli, all growth conditions wereidentical. One experiment representative of three (each performed with adifferent isolate).

[0027]FIG. 3 Gastrin uptake assay.

[0028]H. pylori (H.P.), E. coli (E.C.), S. pneumonia (S.P.) and C.jejuni (C.J.) bacteria were incubated for 45 minutes with ¹²⁵I-labeledgastrin. Incubation was at 4° C. or 37° C. Following initial incubation,proteinase K (P.K) was added for 30 minutes, after which bacteria werewashed and blotted. One experiment representative of two.

[0029]FIG. 4 Competition by diferent peptides on ¹²⁵I-labeled gastrinuptake by H. pylori.

[0030] Bacteria were incubated in the presence of 500 nmo/l ¹²⁵I-labeledgastrin and increasing concentrations of either unlabeled gastrin (G),CCK-8 (CCK), pentagastrin (Pen), somatostatin (Som) or EGF for 45 min.Bacteria were washed and radioactivity remaining in bacterial pelletswas determined. One experiment representative of three.

DETAILED DESCRIPTION OF THE INVENTION

[0031]H. pylori infection is associated with duodenal ulcer, gastriculcer, gastric adenocarcinoma and B cell lymphoma. It has also beenimplicated in cardiovascular diseases, particularly atherosclerosis andischemia, since it has been shown that the infection is more abundant inpatients suffering from these diseases. The bacterium resides within thegastric mucosa, a unique niche hostile to other microorganisms. Gastrin,a gastric hormone, is consistently elevated following H. pyloriinfection. It has now been found that human gastrin stimulates H. pylorigrowth in a specific, dose-dependent mechanism, which suggests a novelapproach to treating H. pylori infections.

[0032] As demonstrated in the following Examples, the human hormonegastrin significantly stimulated H. pylori growth in a dose-dependentmanner. This effect was reproducible in all bacterial isolates studied.Growth stimulation occurred at physiological gastrin concentrationsfound in the blood and gastric lumen [Mueller C R, et al., Surgery(1991) 110:1116-1124; Yamashita K, et al., Gastroenterology (1998)115:1123-1130].

[0033] The uptake of gastrin by H. pylori and the stimulatory growtheffect were highly specific and were not detected with control bacteriaor by other peptides present in the gastric antrum or lumen. Moreover,cold ligand inhibition suggested the effect was mediated via a specificgastrin binding site. Gastrin, CCK-8 and pentagastrin have structuralsimilarity and activate human gastrin/CCKB receptors. In the presentexperiments, both CCK-8 and pentagastrin competed with gastrin on thebinding to H. pylori, but did not stimulate bacterial growth. Thecompetition by structurally related compounds supports the suggestionthat a specific binding site is involved in gastrin uptake by H. pylori.The fact that biological activity could be obtained only with gastrin,reiterates the specific interaction of H. pylori with this hormone.

[0034] In light of the present findings, host parasite interactionbetween the human host and H. pylori could be viewed as having evolvedto exploit gastrin by the bacterium. H. pylori preferentially colonizesthe gastric antrum, the anatomic location of gastrin producing G cells[Bayerdorffer E, et al., Gastroenterology (1992) 102:1575-1582]. Inaddition, infection results in hypergastrinemia and a loss of negativefeedback control on gastrin secretion, potentially leading to thecontinuous exposure of bacteria to high levels of gastrin.

[0035] The histologic hallmark of H. pylori infection is chronic antralinflammation. As a result of this inflammation, increased levels ofpro-inflammatory cytokines have been documented [Crabtree J E, et al.,Gut (1991) 32:1473-1477; Karttunen R, et al., Gut (1995) 36:341-345;Noach L A, et al., Scand J Gastroenterol (1994) 29:425-429; Crabtree JE, et al., Scand J Immunol (1993) 37:65-70]. Using in vitro cultures, itwas shown that endocrine cells can produce gastrin in response tostimulation with such inflammatory cytokines [Lehmann F S, et al., Am JPhysiol (1996) 270:783-788; Weigert N, et al., Gastroenterology (1996)110:147-154; Beales I L, et al., Eur J Gastroenterol Hepatol (1997)9:773-777; Wallace J L, et al Am J Physiol (1991) 261:G559-564]. Thus,H. pylori may actually benefit from the host immune response, whichinitiates up-regulation of a bacterial growth factor.

[0036] Models based on H. pylori characteristics and host parasiteinteraction have been proposed. Lee suggested that the exceptionallysmall size of the H. pylori genome and the small number of regulatorygenes might indicate that the bacterium is well adapted to a highlyspecific single habitat [Lee A, N Engl J Med (1998) 338:832-833]. Thefinding that H. pylori utilizes gastrin, a gastric hormone, concurs withthis proposal. Furthermore, the chronicity and persistence of H. pyloriinfection in the human host led Blaser to suggest that the interactionbetween bacterium and host is mutually regulated. Such regulationimplies co-evolution [Blaser M J, J Clin Invest (1997) 100:759-762].Increased gastrin secretion as a consequence of infection and thepossible exploitation of gastrin by the bacterium fit this concept.

[0037] Therapy of peptic ulcer disease prior to the discovery of thecentral role of H. pylori, was based on agents capable of reducinggastric acidity. Based on meta analysis of multiple trails aimed athealing peptic ulcers, it was shown that there is no advantage inelevating the pH to above 3, for more than 18 hours daily. Still,several groups of agents are used for reducing the stomach acidity. Mostimportant of these agents are the H2 receptor antagonists and protonpump inhibitors. H2 receptor antagonists act by blocking histamine H2receptors on the parietal cells. Proton pump inhibitors act byinhibition of the parietal cell H⁺K⁺ ATPase, responsible for acidsecretion from the cells.

[0038] Current therapies of H. pylori infections usually consist ofcombinations of two antibiotic agents together with an adjunctive agent,which is usually either a proton pump inhibitor or bismuth. Antibioticresistance of H. pylori is widespread, with increasing prevalence[Hazell, S L, Eur J Clin Infect Dis (1999) 18:83-86]. H.pylori-associated infection is a rather common phenomenon, resulting ina massive use of antibiotics, sometimes prescribed also to a symptomaticcarriers. This massive use may exhaust the option of treatment of thisdisease by currently available drugs, and the resistance of thebacterium may later adversely affect the extent of success of therapy ofH. pylori infection, as well as other infectious agents that may becomeresistant, as a secondary effect of extensive use of antibiotics.

[0039] The present findings suggest that gastrin may be the factor onwhich H. pylori thrives, and in its absence, the bacterium will not beable to survive in the gastric antrum. The growth benefit provided to H.pylori by gastrin, may be the reason for the ability of this bacteriumto flourish in this hostile habitat.

[0040] It is this newly discovered relationship between gastrin and H.pylori, which provides the basis for alternative, non-antibiotic-basedimproved therapies of various diseases associated with H. pyloriinfection, particularly those of the gastrointestinal tract.

[0041] Thus, the invention relates to a pharmaceutical composition forthe treatment and/or prevention of H. pylori-associated disorderscomprising as active ingredient a therapeutically effective amount of acompound which inhibits the growth-enhancing effect of gastrin on H.pylori, optionally further comprising pharmaceutically acceptablecarriers, adjuvants or diluents.

[0042] The pharmaceutical composition of the invention may beparticularly suitable for the treatment and/or prevention of H.pylori-associated gastrointestinal disorders, specifically, H.pylori-associated gastric and/or duodenal peptic diseases. In additionthe compositions of the invention may be used for the treatment ofgastritis, duodenitis, non-ulcer dyspepsia, mucosal-associated lymphoidtissue lymphoma, for the prevention of gastric carcinoma and in thetreatment of atherosclerotic cardiovascular diseases.

[0043] The active ingredient in the pharmaceutical compositions of theinvention is preferably a compound which is capable of inhibitinggastrin uptake by H. pylori. Such compound may be a competitiveinhibitor of gastrin uptake by H. pylori, or an antagonist of the humanor H. pylori gastrin receptor.

[0044] In preferred embodiments, the active compound comprised in thepharmaceutical composition of the invention is a peptide. Preferredpeptides may be synthetic analogue of gastrin or of a fragment ofgastrin, preferably comprising an amino acid sequence corresponding tothe C-terminal of gastrin or to a gastrin fragment, preferably G17. Suchpreferred peptides comprise the amino acid sequence: Trp-Met-Asp-PheNH₂.Particular examples of synthetic peptides which may be used as theactive principal in the compositions of the invention are pentagastrinor cholecystokinin (CCK)-8.

[0045] Pentagastrin is a synthetic gastrin analogue that exhibits thebiological activity of gastri. The main clinical use of this compoundwas for stimulation of acid secretion in a diagnostic test required forexclusion of achlorhydria (acid stimulation occurs within 10 minutes andpeaks within 20-30 minutes). Pentagastrin replaced the use of histaminein such tests, because it has milder side effects.

[0046] Synthetic peptides used in the compositions of the invention maybe in the form of a dimer, a multimer or in a constrained conformation,in which the constrained conformation is obtained by internal bridges,short-range cyclizations, extension or other chemical modification.

[0047] In a further embodiment, the active compound comprised in thepharmaceutical compositions of the invention may be a non-peptidicantagonist of the human or H. pylori gastrin receptor.

[0048] In a further aspect, the present invention relates to a methodfor the treatment and/or prevention of H. pylori-associated disorders ina patient in need of such treatment, comprising administering to saidpatient a therapeutically effective amount of a compound which inhibitsthe growth-enhancing effect of gastrin on H. pylori or a therapeuticallyeffective amount of a composition according to the invention.

[0049] The method of the invention is particularly suitable for thetreatment and/or prevention of H. pylori-associated gastrointestinaldisorders, but is not limited thereto.

[0050] The compound to be administered by the method of the invention isa compound which capable of inhibiting gastrin uptake by H. pylori.Particular compounds may be competitive inhibitors of gastrin uptake byH. pylori and/or antagonists of the human or H. pylori gastrin receptor.

[0051] In preferred embodiments, the compound to be administered is apeptide, preferably a synthetic analogue of gastrin or of a fragment ofgastrin, preferably of G17. More preferred peptides the amino acidsequence: Trp-Met-Asp-PheNH₂, for example pentagastrin orcholecystokinin (CCK)-8. In other embodiments of the method of theinvention the compound to be administered may be a non-peptidicantagonist of the human or H. pylori gastrin receptor.

[0052] Still further, the invention relates to use of a compound whichinhibits the growth-enhancing effect of gastrin on H. pylori in thepreparation of pharmaceutical compositions for the treatment of H.pylori-associated disorders, particularly in the preparation ofpharmaceutical compositions for the treatment of H. pylori-associatedgastrointestinal disorders.

[0053] All of the compounds referred to above may be used in thepreparation of pharmaceutical compositions according to the use of theinvention.

[0054] The pharmaceutical compositions of the invention will generallycontain salts, preferably in physiological concentration, such as PBS(phosphate-buffered sale), or sodium chloride (0.9% w/v), and abuffering agent, such as phosphate buffer in the above PBS. Thepreparation of pharmaceutical compositions is well known in the art, seee.g., U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378, 5,439,688,5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549, 4,755,383,4,639,435, 4,457,917 and 4,064,236. The active ingredient of thepharmaceutical compositions of the present invention, for example apeptide, or pharmacologically acceptable salts thereof, is preferablymixed with an excipient, carrier, diluent, and optionally, apreservative or the like pharmacologically acceptable vehicles as knownin the art, see e.g., the above U.S. patents. Examples of excipientsinclude, glucose, mannitol, inositol, sucrose, lactose, fructose,starch, corn starch, microcrystalline cellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, polyvinylpyrrolidone and the like.Optionally, a thickener may be added, such as a natural gum, a cellulosederivative, an acrylic or vinyl polymer, or the like.

[0055] The pharmaceutical composition is provided in solid, liquid orsemi-solid form. A solid preparation may be prepared by blending theabove components to provide a powdery composition. Alternatively, thepharmaceutical composition is provided as lyophilized preparation. Theliquid preparation is provided preferably as aqueous solution, aqueoussuspension, oil suspension or microcapsule composition. A semi-solidcomposition is provided preferably as hydrous or oily gel or ointment.About 0.001 to 60 w/v %, preferably about 0.05 to 25 w/v % of the activeagent is provided in the composition.

[0056] A solid composition may be prepared by mixing an excipient with asolution of the active agent comprised in the composition of theinvention, gradually adding a small quantity of water, and kneading themixture. After drying, preferably in vacuo, the mixture is pulverized. Aliquid composition may be prepared by dissolving, suspending oremulsifying the active compound in water, a buffer solution or the like.An oil suspension may be prepared by suspending or emulsifying theactive compound in an oleaginous base, such as sesame oil, olive oil,corn oil, soybean oil, cottonseed oil, peanut oil, lanolin, petroleumjelly, paraffin, Isopar, silicone oil, fatty acids of 6 to 30 carbonatoms or the corresponding glycerol or alcohol esters. Buffers includeSorensen buffer [Ergeb Physiol, (1912) 12:393], Clark-Lubs buffer [JBact (1917) 2(1):109 and 191]. MacIIvaine buffer [J Biol Chem (1921)49:183], Michaelis buffer (Die Wasserstoffinonenkonzentration, p. 186,1914), and Kolthoff buffer [Biochem Z, (1926) 179:410].

[0057] A composition may be prepared as a hydrous gel, e.g. fortransnasal administration. A hydrous gel base is dissolved or dispersedin aqueous solution containing a buffer, and the said active agent, andthe solution warmed or cooled to give a stable gel.

[0058] Preferably, the composition of the invention is administeredthrough intravenous, intramuscular or subcutaneous administration. Oraladministration is expected to be less effective, particularly where theactive compound is a peptide, because the peptide may be digested beforebeing taken up. Of course, this consideration may apply less to anactive peptide invention which is modified as described above, e.g., bybeing cyclic peptide, by containing non-naturally occurring amino acids,such as D-amino acids, or other modification which enhance theresistance of the peptide to biodegradation. Decomposition in thedigestive tract may be lessened by use of certain compositions, forinstance, by confining the active compounds comprised in thecompositions of the invention in microcapsules such as liposomes. Thepharmaceutical composition of the invention may also be administered toother mucous membranes. The pharmaceutical composition is then providedin the form of a suppository, nasal spray or sublingual tablet. Thedosage of the peptide of the invention may depend upon the condition tobe treated, the patient's age, body weight, and the route ofadministration, and will be determined by the attending physician. Dosesof active agent ranging from 0.1 μg/kg to 100 mg/kg or higher,preferably from 0.5 μg/kg to 5 mg/kg, more preferably 0.1 μg/kg to 1mg/kg, most preferably about 100 μg/kg.

[0059] The uptake of an active agent comprised in the composition of theinvention, e.g. an active peptide, may be facilitated by a number ofmethods. For instance, a non-toxic derivative of the cholera toxin Bsubunit, or of the structurally related subunit B of the heal-labileenterotoxin of enterotoxic E. coli may be added to the composition, seeU.S. Pat. No. 5,554,378.

[0060] Alternatively, the pharmaceutical composition of the inventionmay comprise a biodegradable polymer selected from poly-1,4-butylenesuccinate, poly-2,3-butylene succinate, poly-1,4-butylene fumarate andpoly-2,3-butylene succinate, incorporating the active compound, as thepamoate, tannate, stearate or palmitate thereof. Such compositions aredescribed e.g., in U.S. Pat. No. 5,439,688.

[0061] Additionally, a composition of the invention may be a fatemulsion. The fat emulsion may be prepared by adding to a fat or oilabout 0.1-2.4 w/w of emulsifier such as a phospholipid, an emulsifyingaid, a stabilizer, mixing mechanically, aided by heating and/or removingsolvents, adding water and isotonic agent, and optionally, adjustingadding the pH agent, isotonic agent. The mixture is then homogenized.Preferably, such fat emulsions contain an electric charge adjustingagent, such as acidic phospholipids, fatty acids, bilic acids, and saltstherof. Acidic phospholipids include phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. Bilicacids include deoxycholic acid, and taurocholic acid. The preparation ofsuch pharmaceutical compositions is described in U.S. Pat. No.5,733,877.

[0062] The invention will now be described in more detail on hand of thefollowing Examples, which are illustrative only and do not in any senselimit the scope of invention, which is defined by the appended claims.

EXAMPLES

[0063] Materials and Methods

[0064] Bacterial Cultures and Growth Analysis

[0065] Bacteria were obtained from clinical isolates cultured as part ofthe routine work up for H. pylori detection. No bacteria werespecifically obtained for the study. Growth curve analysis was performedusing five different isolates. Endoscopic diagnosis included both pepticpathologies and normal appearing mucosa. Following endoscopy, biopsieswere kept in balanced salt solution for no more than one hour.Subsequently, biopsy specimens were minced and plated on chocolate orColombia agar plats. To generate microaerophilic conditions, bacteriawere grown in sealed jars, using anaerobic gas generating kits (Oxoid,BR-38) at 37° C. Bacteria were re-plated under similar conditions every2-4 days. For experimentation, bacteria were transferred to brain-heartinfusion or Brucella broth liquid media (Gibco), supplemented with 10%calf serum (Biological Industries, Beit Haemek, Israel). Each sample wasinoculated with 0.02 OD 600 corresponding to 1.6×10³ CFU/100 μL. Gastrinor control peptides were added at different concentrations as indicated.

[0066]H. pylori growth necessitates microaerophilic conditions. Repeatedsampling from the same tube would result in disruption of suchconditions. To overcome this problem, in each experiment, multiplesamples in the number of time points assessed were used. Each samplecontained identical bacterial numbers and gastrin concentrations. Adifferent sample was assessed at each time point. Growth was assessed byOD readings.

[0067] All bacteria were obtained from clinical isolates. Growth kineticexperiments with C. jejuni were done in similar conditions as describedabove for H. pylori. Growth kinetic experiments with E. coli, wereperformed under conditions similar to the above, in an aerobic ambient.

[0068] Gastrin and Control Peptides

[0069] Gastrin 17, cholecystokinin (CCK)-8 (fragment 26-33),pentagastrin, somatostatin 14 and epidermal growth factor (EGF) wereobtained from Sigma (St. Louis, Mo.). Lyophilized peptides weredissolved in a stock solution containing acetic acid and water in aratio of 1:1 to a concentration of 1 mg/ml, as suggested by themanufacturer and diluted with sterile deionized water to the indicatedconcentrations.

[0070] Radioactive Gastrin Uptake

[0071] To assess labeled gastrin uptake, bacterial pellets wereresuspended in 200 μl Brucella broth containing 500 nmol/l ¹²⁵I-labeledgastrin (IncStar Pharmatrade). Incubation was at either 4° C. or 37° C.for 45 minutes. Bacteria were washed three times in PBS, resuspended andincubated for 30 minutes in Brucella broth containing proteinase K(Promega) at a concentration of 25 ng/ml. Subsequently, bacteria werewashed, resuspended in 100 μl PBS, out of which 10 μl were blotted onnitrocellulose filters and autoradiography performed for 7 days.Incorporated ¹²⁵I was quantitated using a gamma counter.

[0072] Cold Inhibition Assay

[0073] For the cold inhibition assay, bacterial pellets were resuspendedin 200 μl Brucella broth containing 500 nmol/l ¹²⁵I-labeled gastrin.Unlabeled gastrin in increasing concentrations (1250-5000 nmol/l) wasadded concurrently. Bacteria were incubated for 45 minutes at 37° C.,after which they were extensively washed with PBS. Incorporation of¹²⁵I-labeled gastrin was determined using a gamma counter. As controls,bacteria were incubated with ¹²⁵I-labeled gastrin (500 nmol/l) in thepresence of CCK-8, pentagastrin, somatostatin or EGF at the indicatedconcentrations.

[0074] Results

[0075] To assess the effect of G17 on H. pylori growth, bacteria weregrown with increasing gastrin concentrations. Addition of gastrin to thegrowth media had a dose-dependent effect on the growth curve. Gastrinshortened the lag time, increased the growth rate at the logarithmicphase and increased the final cell density at the stationary phase (FIG.1A). FIG. 1B shows the growth of 5 different clinical isolates in thepresence of gastrin at the 0 and 48h time points. As shown, a similareffect of gastrin was noted for all five isolates.

[0076] To test the specificity of the gastrin effect, growth kineticstudies were done using a slow growing intestinal microaerophihicbacterium and a Gram-negative aerobic intestinal bacterium. As shown inFIGS. 2A-B, gastrin had no effect on the growth of Campylobacter jejuni,or Escherichia coli. To test whether other peptides might also have apositive growth effect similar to gastrin on H. pylori, H. pylori wasgrown in the presence of somatostatin, that is produced in the gastricantrum, and EGF, a growth factor peptide found within the gastric lumen.In addition, since in humans the gastrin and CCKB receptors arehomologous, the gastrin/CCKB receptor agonists CCK-8 and pentagastrin,which have structural homology to gastrin, were tested for their effecton H. pylori growth. As shown in FIGS. 2C-F, none of the peptides had aneffect on the growth of H. pylori, indicating that the positive growtheffect of gastrin was specific.

[0077] To learn whether the uptake of gastrin to H. pylori was specificand whether gastrin was internalized by H. pylori or bound to the outersurface, H. pylori and control bacteria were incubated with ¹²⁵I-labeledgastrin, at 4° C., or 37° C. Following this initial incubation, bacteriawere co-incubated with proteinase K, to digest surface proteins. Labeledgastrin incorporation was assessed by autoradiography and CPMdetermination. As shown in FIG. 3, the uptake of gastrin by the bacteriawas specific for H. pylori, since no labeling was noted with controlbacteria. Further, only H. pylori incubated at 37° C. incorporated the¹²⁵I-labeled gastrin. Such temperature-dependence suggests that anenergy-dependent mechanism is responsible for the uptake of gastrin.Moreover, no reduction in radioactive counts was noted followingco-incubation with proteinase K (before proteinase K incubation—311 CPM,following incubation with proteinase K—385 CPM), suggesting that thegastrin entered the bacteria and thus, was protected from proteinase Kdigestion.

[0078] To test whether gastrin entered the bacteria by a non-specificmechanism or, by interaction with a specific binding site, uptake oflabeled gastrin was competed, using unlabeled gastrin and controlpeptides. As shown in FIG. 4, the uptake of labeled gastrin by H. pyloricould be inhibited with an excess of unlabeled gastrin. Similar tounlabeled gastrin, co-incubation of bacteria in the presence of CCK-8and pentagastrin inhibited labeled gastrin uptake in a dose-dependentmanner. In contrast, no inhibition of gastrin uptake was shown by EGF orsomatostatin.

[0079] The cold-ligand competition and inhibition of gastrin uptake bypeptides known to activate shared receptors in humans, the temperatureprofile of gastrin binding and the protection against proteinase Kdigestion, all suggest the internal localization of a putative receptoror channel with specific structural restriction.

1. A pharmaceutical composition for the treatment and/or prevention ofH. pylori-associated disorders comprising as active ingredient atherapeutically effective amount of a compound which inhibits thegrowth-enhancing effect of gastrin on H. pylori, optionally furthercomprising pharmaceutically acceptable carriers, adjuvants or diluents.2. A pharmaceutical composition according to claim 1 , for the treatmentand/or prevention of H. pylori-associated gastrointestinal disorders. 3.A pharmaceutical composition according to claim 1 or claim 2 , whereinsaid compound is capable of inhibiting gastrin uptake by H. pylori.
 4. Apharmaceutical composition according to any one of claims 1 to 3 ,wherein said compound is a competitive inhibitor of gastrin uptake by H.pylori.
 5. A pharmaceutical composition according to any one of claims 1to 3 , wherein said compound is an antagonist of the human or H. pylorigastrin receptor.
 6. A pharmaceutical composition according to any oneof claims 1 to 5 , wherein said compound is a peptide.
 7. Apharmaceutical composition according to claim 6 , wherein said peptideis a synthetic analogue of gastrin or of a fragment of gastrin,preferably of G17.
 8. A pharmaceutical compound according to claim 7 ,wherein said peptide comprises the amino acid sequence:Trp-Met-Asp-PheNH₂.
 9. A pharmaceutical composition according to claim 8, wherein said peptide is pentagastrin or cholecystokinin (CCK)-8.
 10. Apharmaceutical composition according to claim 5 , wherein said compoundis a non-peptidic antagonist of the human or H. pylori gastrin receptor.11. A pharmaceutical composition according to any one of claims 1 to 10, for the treatment and/or prevention of H. pylori-associated gastricand/or duodenal peptic diseases.
 12. A method for the treatment and/orprevention of H. pylori-associated disorders in a patient in need ofsuch treatment, comprising administering to said patient atherapeutically effective amount of a compound which inhibits thegrowth-enhancing effect of gastrin on H. pylori. or a therapeuticallyeffective amount of a composition according to any one of claims 1 to
 11. 13. A method according to claim 12 , for the treatment and/orprevention of H. pylori-associated gastrointestinal disorders.
 14. Amethod according to claim 13 or 14 , wherein said compound is capable ofinhibiting gastrin uptake by H. pylori.
 15. A method according to claim14 , wherein said compound is a competitive inhibitor of gastrin uptakeby H. pylori.
 16. A method according to claim 12 or 13 , wherein saidcompound is an antagonist of the human or H. pylori gastrin receptor.17. A method according to any one of claims 12 to 16 , wherein saidcompound is a peptide.
 18. A method according to claim 17 , wherein saidpeptide is a synthetic analogue of gastrin or of a fragment of gastrin.19. A method according to claim 18 , wherein said peptide is a syntheticanalogue of G17.
 20. A method according to claim 19 , wherein saidpeptide comprises the amino acid sequence: Trp-Met-Asp-PheNH₂.
 21. Amethod according to claim 20 , wherein said peptide is pentagastrin orcholecystokinin (CCK)-8.
 22. A method according to any one of claims 12to 16 , wherein said compound is a non-peptidic antagonist of the humanor H. pylori gastrin receptor.
 23. Use of a compound which inhibits thegrowth-enhancing effect of gastrin on H. pylori in the preparation of apharmaceutical composition for the treatment of H. pylori-associateddisorders.
 24. Use according to claim 23 , in the preparation of apharmaceutical composition for the treatment of H. pylori-associatedgastrointestinal disorders.
 25. Use according to claim 23 , wherein saidcompound is a synthetic analogue of G17, preferably comprising the aminoacid sequence: Trp-Met-Asp-PheNH₂.
 26. Use according to claim 23 ,wherein said compound is a non-peptidic antagonist of the human or H.pylori gastrin receptor.